Fig 2: GABA levels are positively correlated with GRP levels in patients with NE markers. (a) A positive correlation between GABA and GRP levels was found in PCa patients (r = 0.894; p = 0004; n = 10), and when patients were separated, PCa-NE patients had a strong positive correlation (r = 0.961; p = 0.03; n = 4). (b–d) Relative AR, ENO2 (*p < 0.05), GABBR1 (**p < 0.001) gene expression in BPH vs. PCa and BPH vs. PCa-NE, respectively. AR mRNA levels were not significantly different between groups. (e) PCA3 mRNA levels were significantly higher in PCa patients compared to BPH patients (*p < 0.05).

Fig 3: Establishment of NE-like cell line by siRNA targeted to the AR silence in LNCaP cell. (a) AR mRNA expression levels were analyzed by RT-qPCR in LNCaP cells transfected with AR-specific siRNA and control siRNA from 24 h to 96 h in RPMI supplemented with 10% FBS. Experiments were repeated four times. Differences among groups were compared by two-tailed unpaired Student’s t test, *p < 0.0001. (b) Upper panel, Representative Western blot analysis of AR, ENO2 and GRP expression in LNCaP cells transfected with AR-specific siRNA and Control siRNA from 24 h to 96 h. GPDH was used as a loading control. Lower panel, ImageJ software was used for densitometric quantification of proteins. Graphs represent the mean ± SEM (n = 3) *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control group. The samples derive from the same experiment and that gels/blots were processed in parallel. Full-length blots/gels are presented in Supplementary Fig. 1.

Fig 4: GABA promotes GRP secretion mediated by GABBR1 in NE-like cells. (a) GABBR1 mRNA expression was evaluated by RT-qPCR of 96 h AR siRNA cells and 96 h control siRNA cells grown for 12 h in RPMI with indicated concentrations of baclofen. Differences among groups were compared by two-tailed unpaired Student’s t test, *p < 0.0001. (b) GABBR1 protein expression was evaluated by Western Blot in whole cell lysate. GAPDH was used as a loading control. Graphs represent the mean ± SEM (n = 4), *p < 0.001. The last result was normalized to 1 with respect to the vehicle. The concentrations of GRP in the medium of 96 h AR siRNA cells and 96 h control siRNA cells were examined by ELISA (n = 3/group). (c) GRP was present in the medium of both cells incubated with RPMI containing different concentrations of Baclofen (GABAB agonist) 1, 10 and 100 µM for 12 h. Differences among groups were compared by two-tailed unpaired Student’s t test, *p < 0.001 and **p < 0.0001. (d) GABAB antagonist (CGP 35348) was used, and cells were treated with 10, 50 and 100 µM in combination with baclofen (100 µm) for 12 h. Differences among groups were compared by two-tailed unpaired Student’s t test, *p < 0.001 and **p < 0.0001. The samples derive from the same experiment and that gels/blots were processed in parallel. Full-length blots/gels are presented in Supplementary Fig. 1.